Diagnostic agent for the detection of peroxidatively active substances

ABSTRACT

Test strips for the detection of peroxidatively active substances in body fluids, e.g., for the detection of blood in urine, comprising a carrier containing A. A HYDROPEROXIDE; B. AT LEAST ONE CHROMOGEN; AND C. AS AN ACTIVATOR A COMPOUND OF THE FORMULA:   WHEREIN R1 is pyridyl or phenyl, optionally substituted by lower alkyl or alkoxy, and R2 is hydrogen or lower alkyl.

' United States Patent 1 Rittersdorf et al.

[ 1 Nov. 4, 1975 [73] Assignee: Boehringer Mannheim GmbH,

Mannheim, Germany 22 Filed: Nov. 25, 1974 21 Appl. No.: 526,661

[30] Foreign Application Priority Data Dec. 20, 1973 Germany 2363344 [52] US. Cl. 23/230 B; 23/253 TP; 195/103.5;

[51] Int. CL? .,C07D 213/24; GOIN 31/22; GOIN 33/16 [58] Field of Search 23/230 B, 253 TP; 195/103.5; 252/408; 260/240 D, 240 E [56] References Cited UNITED STATES PATENTS 3,558,435 1/1971 Rey et a1. .4 23/230 B X 3,630,847 12/1971 Rey et a1 23/230 B X 3,654.179 4/1972 Bauer i 23/230 B X 3,654.180 4/1972 Bauer 23/230 B X 3.853.471 12/1974 Rittersdorf et al. 23/230 B Primary Examiner-Robert M. Reese Attorney, Agent, or FirmBurgess, Dinklage & Sprung [57] ABSTRACT Test strips for the detection of peroxidatively active substances in body fluids, e.g., for the detection of blood in urine, comprising a carrier containing a. a hydroperoxide;

b. at least one chromogen; and

c. as an activator a compound of the formula:

wherein R is pyridyl or phenyl, optionally substituted by lower alkyl or alkoxy, and R is hydrogen or lower alkyl.

25 Claims, No Drawings DIAGNOSTIC AGENT FOR THE DETECTION OF PEROXIDATIVELY ACTIVE SUBSTANCES The present invention relates to a test strip for the de tection of very small amounts of blood and of other peroxidatively-active substances in the body fluids.

The detection of small amounts of blood, which cannot be seen by the naked eye, in urine, feces and vomit is very important for the diagnosis of hemorrhages in the stomach, intestines and urinary tract. Such hemorrhages are caused, for example, by tumors, ulcers or inflammations in the organs in question. Furthermore, due to the .influence of certain hemolytic toxins, free hemoglobin can also occur in the urine and plasma. Blood and hemoglobin are .peroxidatively-active, i.e., they liberate oxygen from hydroperoxides and transfer it to certain acceptors. Myoglobin, which is also peroxidatively-active, is found, for example, in the urine after a cardiac infarct. Furthermore, blood occurs especially frequently in the urine when calculi are present in the bladder or kidneys. I

For asensitive detection of all of these substances, their peroxidate action is especially suitable. The oxygen liberated from a hydroperoxide is thereby transmitted to a chromogen which is oxidized to a color mate rial and thus indicates the presence of the peroxidative ly-active substances. This reaction has been used for quite a long time in medicinal and forensic analysis, especially for the detection of blood. As a rule, the reaction is carried out in a test tube or as a spot test, hydrogen peroxide usually being employed as the hydroper oxide. As chromogen, there is usually employed benzidine, o-tolidine or leucomalachite green.

Rapid test compostions are absorbent carriers, usually papers, which are impregnated with all the reagents needed for the detection reaction and which, after simply dipping into the body fluid to be tested, show a color reaction. Because of the great importance which these rapid tests have recently achieved, they have also been developed in various ways for the detection of blood in body fluids.

Since, for the detection of blood, the sensitivity of the rapid test is of decisive importance, various attempts have already been made to increase the sensitivity of the known detection reactions by means of additives. Thus, for example, German Pat. Spec. No. 1,242,905 describes test papers which, as activating additives, contain certain quinoline derivatives, preferably quinine. It is certainly asserted that the sensitivity can be considerably increased by means of these additives but, according to our own investigations, it is practically impossible, with the so improved tests strips, to detect, for example, blood in a concentration of 5 erythrocytes /mm", which would be necessary for a differentiation between normal and pathological values.

We have now found that test papers for the detection of peroxidatively-active substances with a previously unachievably high sensitivity are obtained by utilizing certain vinylpyridine compounds as activators.

The vinyl pyridine compounds showing this efficacious action are of the formula wherein R is pyridyl or phenyl, optionally substituted by lower alkyl or alkoxy, and

R is hydrogen or lower alkyl.

Preferred compounds of general formula (I) are those in which R, is hydrogen. The lower alkyl and alkoxy radicals can contain up to 4 carbon atoms, the methyl and methoxy radicals being preferred.

Thus, according to the present invention, there is provided a test strip for the detection of peroxidativelyactive substances in body fluids, comprising a carrier containing a hydroperoxide, at least one chromogen and, as activator, at least one compound of general formula (I).

The sensitivity of the detection reaction is increased by some of the compounds of general formula (I) to such an extent that it is even possible to detect individual erythrocytes in urine, which are visible on the test paper as colored dots. Thus, for example, with the use of a-styryl-pyridine, it is possible to produce a test paper with which it is possible to detect 5 erythrocytes/mm urine. This corresponds to a blood dilution of 1110.

Of course, not all of the compounds of general formula (I) possess activating properties of the same degree. Thus, it is possible to adjust the sensitivity of, for example, a blood test in accordance with practical requirements. For example, test strips of increasing activity are obtained when, as activator, there are used the following compounds in the given order:

2-( phenylvinyl )-4-methylpyridine Pyridyl-Z-pyridyl-4-ethylene 2-( methylphenylvinyl)-pyridilne 2-(4-methoxyphenylvinyl) pyridine di-(pyridyl-2)-ethylene pyridyl-2-pyridyl-3-ethylene oz-styryl-pyridine.

The activating agent used according to the present invention can be employed in amounts of about 0.05 1.0g, preferably of 0.2 0.5g, per ml. ofimpregnation solution.

Further components of a rapid test for blood are an organic hydroperoxide, an oxidation indicator (chromogen), a buffer and a surface-active agent, as well as, if desired, a phosphoramide, for example phosphoric acid trimorpholide, for stabilization, as well as conventional adjuvants.

As hydroperoxides, there can be used, for example, diisopropyl-benzene hydroperoxide or 2,5-dimethylhexane-2,S-dihydroperoxide, and as indictors, there can be used, for example, those of the benzidine series, such as o-tolidine, or those of the heterocylic azine series, such as bis-(N-ethyl-quinol-Z-one)-azine or (N- methyl-benz-thiazol-2-one)-(1-ethyl-3-phenyl-5- methyltriazol-Z-one)-azine (see German Pat.Spec No. 1,648,840).

The indicator (chromogen) can be used in amounts of from 0.05 g., preferably of 0.2 1.0 g., per 100 ml. impregnation solution.

As buffer, there can be used, for example, a citrate. phosphate, phthalate or succinate buffer. the pH value and capacity being so chosen that. after dipping the test strip into a body fluid, a pH value of 4 7. preferably 5 3 6, is obtained thereon.

it is also advantageous to add to the formulation small amounts (for example about 0.05 0.5 g. per 100 ml.) ofa complex-forming agent, for example, sodium metaphosphate or ,ethylenediamine-tetraacetic acid, in order to avoid falsely positive reactions which could be caused bytraces of metals.

Since the test papers can, because of the relatively large amounts 7 of water-soluble substances present, tend to bleed, it is practical to add to the formulation a thickening agent, for example, methyl cellulose and, in particular, polyvinylpyrrolidone, preferably in amounts of about 0.5 5 g. per 100 ml. g I

wetting agent, there is preferably used a longchainedorganic sulphate or sulphonate, for example sodium 'dodecyl-benzenesulphonate, dioctyl sodium sulphosuccinate or sodium lauryl sulphate, which, as is known, stabilizes radical cations, such as oxidized otolidine. The wetting agent can be added to the impregnation solutionin amounts of from 0.5 5%, preferably of'l 3%. I

For the production of the test strips according to the present invention, absorbentcarriers, for example, filter paper, cellulose or synthetic fibre fleeces, can be impregnated with solutions of the reagents'in readily volatile liquids. This is preferably carried out in two or three separate steps. Thus, for example, impregnation is first carried out with a solution which contains a hydroperoxide, wetting agent, buffer and optionally a thickening agent. Thereafter, impregnation is carried out witha solution of an indicator and of an activator of general formula (1).

After drying, the test strips according to the present invention are cut up into strips and preferably sealed between a synthetic resin film and a fine-mesh material in the manner described in German Pat. Spec. No. 2,118,455.

For the detection of peroxidatively-active substances in feces, it is also possible to incorporate the activators according to the present invention, together with the reagents, in a water-stable film in the manner described in German Pat. Spec. No. 1,598,153. This has the advantage that the surface of the test strip can, for readingoff the color reaction, be cleaned by simply wiping it.

The following Examples are given for the purposes of illustrating the present invention:

g, EXAMPLE 1:

Filter paper is successively impregnated with the following solutions and dried'at' 40C.:

Solution 1 152M citrate buffer of pH 5.25

-con tinued g, v

distilled water ad l00.0 ml.

Solution 2.

o-tolidine 0.3 g. o-stilhazole 0.2 g. toluene ad l00.0 ml.

A'white test paper is obtained which, upon dipping into blood-containing urine, becomes blue-green colored after about 5 20 seconds. If the erythrocytes are intact, then the papers are blue-green speckled. If hemolysis has taken place or if free hemoglobin was initially present in the urine, then the papers are uniformly blue-green colored. The sensitivity is about 5 erythrocytes/mm or the corresponding amount of hemoglobin. A lower number of intact'er'ythrocytes can, under certain circumstances, still bring about'individual green dots on the test paper. The sensitivity with regard to myoglobin corresponds to that for hemoglobin.

Example 2:

Filter paper is impregnated with the followingsolutions and dried at 40C.:

Solution 1 Test papers are obtained with'practically the same properties as those described in Example I. The sensitivety is between about 5 and 20 erythrocytes/mm .'de-

creasing with the compound (I) used in the given order:

Compound I used:

pyridyl-2-pyridyl-3-ethylene di-(Pyridyl-Z)-ethylene 2-(4-methoxyphenylvinyl)-pyridine 2-(4-methylphenylvinyll-pyridine pyridyl-2-pyridyl-4-ethylene -continucd 2-( phenylvinyl 4-methylpyridine.

All the abovementioned compounds are known from the literature.

It will be understood that the foregoing specification and examples are illustrative but not limitative of the present invention inasmuch as other embodiments within the spirit and scope of the invention will suggest themselves to those skilled in the art.

What is claimed is:

1. Test strip composition for the detection of peroxidatively-active substances in body fluids, comprising a carrier containing a hydroperoxide, at least one chromogen and, as an activator, a compound of the formula:

wherein R is pyridyl or a phenyl optionally substituted by lower alkyl or alkoxy; and

R is hydrogen or lower alkyl.

2. Test strip as claimed in claim 1 and wherein R in the formula of said activator compound is pyridyl.

3. Test strip as claimed in claim 1 wherein R in the formula of said activator compound is phenyl.

4. Test strip as claimed in claim 1 wherein R in the formula of said activator compound is pyridyl substituted by methyl or methoxy.

5. Test strip as claimed in claim 1 wherein R in the formula of said activator compound is phenyl substituted by methyl or methoxy.

6. Test strip as claimed in claim 1 wherein R in the formula of said activator compound is hydrogen.

7. Test strip as claimed in claim 1 wherein R in the formula of said activator compound is methyl.

8. Test strip as claimed in claim 1 wherein said activator compound is Z-(phenylvinyl)-4-methylpyridine.

9. Test strip as claimed in claim 1 wherein said activator compound is pyridyl-2-pyridyl-4-ethylene.

10. Test strip as claimed in claim 1 wherein said activator compound is 2-(4-methylphenylvinyl)-pyridine.

11. Test strip as claimed in claim 1 wherein said activator compound is 2-(4-methoxyphenylvinyl)-pyridine.

12. Test strip as claimed in claim 1 wherein said activator compound is di-(pyridyl-2)-ethylene.

13. Test strip as claimed in claim 1 wherein said activator compound is pyridyl-2-pyridyl-3-ethylene.

14. Test strip as claimed in claim 1 wherein said activator compound is a-styryl-pyridine.

15. Test strip as claimed in claim 1 wherein said hydroperoxide is diisopropyl-benzene hydroperoxide or 2,5dimethylhexane-2,S-dihydroperoxide.

16. Test strip as claimed in claim 1 wherein said indicator is of the benzidine series or of the heterocyclic azine series.

17. Test strip as claimed in claim 1 wherein said composition also contains a small amount of a complex forming agent.

18. Test strip as claimed in claim 1 wherein said composition also contains a thickening agent.

19. Test strip as claimed in claim 1 wherein said composition also contains a wetting agent.

20. Test strip as claimed in claim 1 comprising citrate buffer ethylenediaminetetraacetic: acid, disodium salt dodecyl-benzene-sulfonic acid sodium salt 2,S-dimethylhexane-Z.S-dihydroperoxide phosphoric acid trimorpholide o-tolidine o-stilbazole.

21. Test strip as claimed in claim 1 wherein the carrier is an absorbent material impregnated with the reagents.

22. Test strip as claimed in claim 21 wherein the carrier is a water-stable film with the reagents incorporated therein.

23. Method of detecting a peroxidatively active substance in a body fluid which method comprises contacting a sample of said body fluid with a test strip composition as claimed in claim 1 and observing a color change in said test strip composition as an indication of the absence or presence of the peroxidatively active sub stance.

24. Method as claimed in claim 23 wherein said peroxidatively active substance is blood.

25. Method as claimed in claim 23 wherein said body fluid is urine. 

1. TEST STRIP COMPOSITION FOR THE DETECTION OF PEROXIDATIVELY-ACTIVE SUBSTANCES IN BODY FLUIDS, COMPRISING A CARRIER CONTAINING A HYDROPEROXIDE, AT LEAST ONE CHROMOGEN AND, AS AN ACTIVATOR, A COMPOUND OF THE FORMULA:
 2. Test strip as claimed in claim 1 and wherein R1 in the formula of said activator compound is pyridyl.
 3. Test strip as claimed in claim 1 wherein R1 in the formula of said activator compound is phenyl.
 4. Test strip as claimed in claim 1 wherein R1 in the formula of said activator compound is pyridyl substituted by methyl or methoxy.
 5. Test strip as claimed in claim 1 wherein R1 in the formula of said activator compound is phenyl substituted by methyl or methoxy.
 6. Test strip as claimed in claim 1 wherein R2 in the formula of said activator compound is hydrogen.
 7. Test strip as claimed in claim 1 wherein R2 in the formula of said activator compound is methyl.
 8. Test strip as claimed in claim 1 wherein said activator compound is 2-(phenylvinyl)-4-methylpyridine.
 9. Test strip as claimed in claim 1 wherein said activator compound is pyridyl-2-pyridyl-4-ethylene.
 10. Test strip as claimed in claim 1 wherein said activator compound is 2-(4-methylphenylvinyl)-pyridine.
 11. Test strip as claimed in claim 1 wherein said activator compound is 2-(4-methoxyphenylvinyl)-pyridine.
 12. Test strip as claimed in claim 1 wherein said activator compound is di-(pyridyl-2)-ethylene.
 13. Test strip as claimed in claim 1 wherein said activator compound is pyridyl-2-pyridyl-3-ethylene.
 14. Test strip as claimed in claim 1 wherein said activator compound is Alpha -styryl-pyridine.
 15. Test strip as claimed in claim 1 wherein said hydroperoxide is diisopropyl-benzene hydroperoxide or 2,5-dimethylhexane-2,5-dihydroperoxide.
 16. Test strip as claimed in claim 1 wherein said indicator is of the benzidine series or of the heterocyclic azine series.
 17. Test strip as claimed in claim 1 wherein said composition also contains a small amount of a complex forming agent.
 18. Test strip as claimed in claim 1 wherein said composition also contains a thickening agent.
 19. Test strip as claimed in claim 1 wherein said composition also contains a wetting agent.
 20. Test strip as claimed in claim 1 comprising citrate buffer ethyLenediamine-tetraacetic acid, disodium salt dodecyl-benzene-sulfonic acid sodium salt 2,5-dimethylhexane-2,5-dihydroperoxide phosphoric acid trimorpholide o-tolidine o-stilbazole.
 21. Test strip as claimed in claim 1 wherein the carrier is an absorbent material impregnated with the reagents.
 22. Test strip as claimed in claim 21 wherein the carrier is a water-stable film with the reagents incorporated therein.
 23. Method of detecting a peroxidatively active substance in a body fluid which method comprises contacting a sample of said body fluid with a test strip composition as claimed in claim 1 and observing a color change in said test strip composition as an indication of the absence or presence of the peroxidatively active substance.
 24. Method as claimed in claim 23 wherein said peroxidatively active substance is blood.
 25. Method as claimed in claim 23 wherein said body fluid is urine. 